Systemic lupus erythematosus is a sometimes fatal autoimmune disease primarily affecting women. Estrogen contributes to disease severity, but does not completely explain the female predilection. Recent reports that men with Klinefelter's Syndrome (XXY) develop lupus at the same rate as women, while women with Turner's Syndrome (XO) are protected, suggests that having 2 X chromosomes is important for disease development. However, how a second X chromosome, which is largely inactivated, contributes to lupus in women is unclear. Evidence accumulated over more than 20 years indicates that lupus develops when genetically predisposed people are exposed to environmental agents that cause DNA demethylation in T cells, and that demethylated female T cells are sufficient to cause lupus-like autoimmunity. Work from the Richardson lab has shown that the B cell costimulatory molecule CD40L, encoded on the X chromosome and known to be overexpressed on lupus T cells, is overexpressed on T cells from women but not men with lupus because the gene on the inactive female X demethylates to cause biallelic expression, while men can only transcribe from one gene. Preliminary evidence suggests that demethylated T cells from women also overexpress other X chromosome genes including the chemokine receptor CXCR3, the signaling regulator OGT, and 6 microRNAs. Based on these observations we hypothesize that demethylation of genes on the inactive X predisposes women to lupus. We will test this hypothesis by: 1. Using high throughput DNA sequencing to compare methylation of CXCR3, OGT, the 6 miRNA genes and other X chromosome genes in CD4+ T cells from healthy men and women, with and without treatment with DNA methylation inhibitors, 2. Using high throughput DNA sequencing to compare methylation of CXCR3, OGT, the 6 miRNA genes and other X chromosome genes in CD4+ T cells from men and women with lupus matched for disease activity, 3. Determining the consequences of OGT overexpression on CD4+ T cell gene expression using transfections, microarrays and immunoblotting, and identify the miRNA target transcripts using Argonaute pulldowns followed by array analyses or sequencing, and 4. Determining if overexpressing CD40L, CXCR3, OGT or X chromosome miRNAs in a recently developed murine lupus model increases the severity of lupus in males to approximate the severity in females. We anticipate that these studies will confirm that women are predisposed to lupus in part because they can overexpress more genes than men, identify the molecules responsible, and thereby identify new therapeutic targets for women with lupus. PUBLIC HEALTH RELEVANCE: According to a recent estimate, SLE afflicts more than 130,000 individuals in the United States. Of these, nearly 90% are women, and women now comprise a significant number of military veterans. Current estimates reveal that the mortality rate of women with lupus is as high as 17.6 per million per year, indicating that current therapies are still imperfect. The design of more effective treatments will require a clear understanding of lupus pathogenesis. The studies described in this applicatiion will characterize molecular mechanisms predisposing women to lupus and thus may contribute to the design of safer and more effective therapies for this disease, and possibly for other autoimmune diseases that afflict our female veterans.